Estriol [estra-1.3.5(10)-triene-3,16.alpha.,17.beta.-triol], an estrogenic hormone, is the predominant steroid hormone most frequently found in the urine of pregnant women. The placenta produces large amounts of this hormone and both serum and urine levels of estriol have been determined in an effort to confirm early stage pregnancy. Monitoring hormone levels continues to indicate the state of fetoplacental function throughout pregnancy. Determinations of 17.beta.-estradiol and estrone [3-hydroxy-estra-1.3.5.(10)-trien-17-one]may assist in evaluation of ovarian function or, in certain circumstances, may be diagnostic for malignancies where normal body functions do not explain increased levels of these hormones. Assays capable of determining levels of estrogenic hormones, therefore, are useful in clinical practice. Moreover, assays which distinguish between estriol and its metabolic precursor 16.alpha.-hydroxyestrone are particularly useful in determination of pregnancy at early stages.
Immunoassays have been recognized as having the speed, sensitivity and convenience necessary to provide an assay for serum estriol at levels in the range of 3-50 ng/mL. Antibodies useful for performing such immunoassays are usually elicited by coupling the desired hapten, such as estriol, to a carrier, most often protein, and injecting these materials into an appropriate host. Estriol contains no functional groups that are themselves amine-reactive. It is necessary, therefore, to use some derivative having an amine-reactive group to couple the derivative to carrier proteins. One such derivative prepared by Dean et al., Steroids Lipids Res., Volume 3, 82-89 (1972). was estriol-6-(0-carboxymethyl)oxime which required a four-step synthetic procedure. This synthesis resulted in an overall yield of about 6%, with the most significant losses occurring in the second reaction in the sequence. This reaction produced multiple side reactions from the oxidation of estriol triacetate to 6-oxoestriol triacetate with chromium trioxide. Necessary purification of the product accounted for additional losses. The need for improved synthetic procedures for estriol derivatives is exemplified by the continued work on improving the existing synthetic routes. Two recent reports of improved methods of forming the 6-oxoestratrienes are Garza et al., Steroids, Volume 42, 469-474 (1983) and Schaumann et al . Ger. (East) DD 200,802 (1983).
U.S. Pat. No. 3,940 475 issued Feb. 24, 1976, discloses another estriol derivative used to prepare antibodies to estriol. 4-(p-carboxyphenylazo)estriol or "capaztriol". It was obtained from the reaction of diazotized p-aminobenzoic acid with estriol under alkaline conditions, followed by chromatographic purification resulting in yields of approximately 15-20%.
A series of estriol and estradiol derivatives was prepared by Walker et al.. Steroids. Volume 21, 259-283 (1973) with substituents at the 6-position. These specific 6-oxo derivatives were used to prepare immunogens with carrier proteins and were compared with randomly linked derivatives for their ability to elicit antibodies with minimal cross-reactivity with structurally similar analogs. It was found that random-linkage immunogens of estriol chloroformates with carrier proteins produced antibodies with 30-100% cross-reactivity, while those with a 6-oxo linked estriol had a 1-12% cross-reactivity with estrone and estradiol. This study suggests that linkage through the 6-position led to immunogens capable of producing antibodies with acceptable specificity However, each derivative required extensive complex synthetic procedures for its preparation, diminishing their convenience and utility in the preparation of immunogens; 6-oxoestriol itself was only produced in 11% yield.
Eshhar et al., protides of the Biological Fluids, Volume 29, 823-826 (1982). used four steroid hormone derivatives to elicit immune responses in C57Bl/6 mice whose spleen cells were then fused with myeloma cells to derive hybridomas. The monoclonal antibodies secreted from these hybridomas were tested for reactivity with several steroid hormones and their derivatives by a coated charcoal radioimmunoassay. Immunogens used were estrone-3-glucuronide-BSA, estradiol-6-(0-carboxymethyl)oxime-BSA. estriol-16.alpha.-glucuronide-BSA and pregnanediol-3.alpha.-glucuronide-BSA. Useful monoclonal antibodies, exhibiting acceptably low cross-reactivity with closely structured analogs, were selected for immunoassays of estrone, estradiol, estriol-16.alpha.-glucuronide, and pregnanediol. Eshhar et al. and the references cited therein do not report yields for the synthesis of estradiol-6-(0-carboxymethyl)oxime. Since, however, this compound was prepared by procedures generally utilized, it is expected that yields similar to the very low yields obtained by Dean et al. were achieved.
Da Re, et al., Arch. Pharm. (Weinheim), Volume 308(12), 981-982 (1975), prepared an estrone derivative that was useful as a therapeutic agent for treatment of high blood pressure and other cardiacrelated conditions. Estrone was reacted with epichlorhydrin under basic conditions in ethanol to produce 3-epoxypropoxy-estra-1.3.5(10)-trien-17-one as an intermediate in 72%. The epoxide was then reacted with isopropylamine in benzene to produce the cardioactive 3-(3'-isopropylamino-2'-hydroxypropoxy)-estra-1.3.5(10)-trien-17-one. It is no known whether the epoxide intermediate might be useful as an immunogen, when coupled with a carrier, to elicit high specificity antibodies.
It is recognized that increased immunoassay specificity can be achieved by using conjugates of low molecular weight materials and carrier proteins as immunogens while utilizing conjugates having different linkages as competitive reagents in the immunoassays. It is believed that the antibodies elicited using one linkage would be less likely to react nonspecifically with conjugates having a second, structurally dissimilar linkage. A reduction in nonspecific reactivity can also be achieved by using different carrier proteins for immunogen and assay reagent.
There is a need for steroid derivative preparations and linking chemistries that will permit rapid, efficient, and economical preparation of conjugates of estriol and estradiol with carrier materials. The conjugates so obtained should be useful in eliciting antibodies specific for the hormone or analog used to prepare them and should also be practical to synthesize.